The present invention relates to new medical uses of lysozyme dimer and to compositions containing such dimer. The new uses are related to treating certain dysfunctions; of natural defensive mechanisms.
Enzymes in their monomeric forms have been known already for a long time to be therapeutically effective in the treatment of various diseases.
Lysozyme was discovered by Fleming in 1922, but it was not before 1950 that its enzymatic functions were revealed. Since that time the compound has been the subject of intensive research and various therapeutic effects were reported. Among others, these were antiviral, antibacterial, anti-inflammatory and antihistaminic properties. The therapeutic use of lysozyme, however, has been rather limited due to the negative side effects of the monomeric form.
This limitation of the practical use of lysozyme and other therapeutically active enzymes was overcome in the late eighties when it was discovered that isolated dimerized forms of enzymes, while retaining all beneficial properties of known monomeric forms, exhibit no negative side effects when used in therapeutic doses. The antiviral and antibacterial compositions comprising as the active ingredient lysozyme dimer or other dimerized enzymes have been described in WO 89/11294. In that application it was reported that in in vitro tests the lysozyme dimer has inhibited proliferation of a number of bacterial strains cultivated on samples taken from patients in concentrations of 5-20 mg/ml of the culture. It was also reported there that the dimer was effective in treating canine parvovirus (CPV) infections when administered orally twice a day at a dose of 1-2 mg/kg of body weight.
As the research work by the inventor was continued, further attractive features of lysozyme dimers were found and new therapeutical uses of the drug were developed.
In clinical tests performed in order to confirm the anti-bacterial and antiviral effectiveness of lysozyme dimer it was surprisingly found out that the dimer is unexpectedly potent in curing acute forms of diseases of the digestive and respiratory tracts. Accordingly, new investigations were carried out in order to determine the effect of lysozyme dimer in those stages of different diseases in which the natural defensive mechanisms fail.
It is known that the bacterial toxins constitute one group of many virulence factors by which bacteria cause diseases. Some recent advances in the knowledge of bacterial toxins concern their interaction with the host's immune system. This interaction firstly results in immunomodulation and secondly in the release of cytokines and other mediators, which account for many physiologic disturbances caused by the toxins. The latter effect has been studied particularly in regard to actions of endotoxin, which plays an important role in the pathogenesis of gram-negative sepsis (see Bayston, D. F., Cohen, J.: Bacterial endotoxins and current concepts in the diagnosis and treatment of endotoxaemia; J. Med. Microbiol. 1990, 31:73-83). Although for long time a role of exotoxins in infections caused by Staphylococcus aureus and Streptococcus pyogenes has been known, it was the recognition of the staphylococcal toxic shock syndrome that led to the increased interest in exotoxins produced by these organisms.
Toxic shock is a severe illness characterized by high fever, hypotension, capillary leak, diffuse erythroderma, mucosal erythema, renal impairment, hypocalcemia, hypoalbuminemia and desquamation of a red skin rash. Many cases of toxic shock syndrome have been associated with the use of vaginal tampons during menstruation but the syndrome is increasingly described in non-menstrual settings in both sexes frequently after surgical procedures when the packing material is left in place (e.g. nasal packings following rhinoplasty or severe epistaxis). The staphylococcus strains isolated from the vagina of patients with toxic shock syndrome (TSS) have been shown to produce toxic shock syndrome toxin 1 (TSST-1), but the source of the microorganism producing TSST-1 may also be an inapparent infection. The initial bacteremia may be inapparent but weeks or months later it may lead to the development of localized infections. Concomitant with the presentation of such infections, there may be evidence of sepsis syndrome or septic shock. A rarer but more dramatic bacteremia may occur in the absence of any portal of entry or associated localized infections, and in these situations shock, endocarditis, disseminated intravascular coagulopathy and multiorgan failure may be prominent (see Stevens, D. L. et al.: Gram-positive shock; Current Opinions in Infectious Diseases 1992, 5:355-363).
Similar observations are known to also involve other gram-positive bacteria. For example Streptococcus pyogenes infection is associated with shock and has a mortality rate of 30%. Streptococcus pyogenes is a cause of pneumonia, which has been documented to be of a high level of resistance to penicillin and tendency of development of a shock syndrome. Moreover, patients with AIDS have a higher incidence of pneumococcal infections than the population as a whole.
Infections with gram-negative bacteria can also result in sepsis and septic shock. Gram-negative bacilli and vibrios are the source of the most important enterotoxins. Enterotoxin is a lipopolysaccharide (LPS) component of the outer membrane of gram-negative bacterial cell walls. Enterotoxins primarily affect the intestinal tract and usually cause diarrhoea. The most frequent infections with gram-negative bacteria among animals and humans are the infections with Escherichia coli. Considerable dehydration accompanying such infections may result in the death of the infected individual. According to WHO, acute diarrhoea kills approximately 3.2 million children in developing countries each year. About 30% of all sepsis cases are caused by gram-negative bacteria.
Sepsis due to infections with gram-positive and gram-negative bacteria is always a severe condition common in all countries. There are approximately 400,000 cases a year within the United States with a mortality rate of approximately 50%.
In recent years, sepsis and septic shock have been a subject of many publications. It has been observed that in the pathophysiology of septic shock, endotoxemia and other bacterial intoxication mediators play the major role. They include tumor necrosis factor (TNF), interleukin-1 (IL-1), interferon (IFN), Platelet-activity factor and eicosanoids (derivatives of arachidonic acid); the most important of these is TNF which has effects on metabolism as well as on the immune and phagocytic systems (see Berkowitz, F. E.: Bacterial toxins in pathogenesis of infections; Current Opinions in Infectious Diseases, 1991 4: 332-337). It was demonstrated that non-survivors of septic shock had higher concentrations of TNF and interleukin-1. Many authors have reported elevated levels of TNF-.alpha. in plasma of the septic shock patients and in their blood. It is pointed also out that the toxic effect of TNF-.alpha. may not depend as much on TNF concentrations as on its persistence within the body.
Many authors have investigated the possibility of modulation of cytokine cascade in sepsis and septic shock. The reported successful proposals involve use of monoclonal anti-TNF antibodies and neutralization of lipopolysaccharide with antilipopolysaccharide. The antibodies however do not enhance the bacterial clearance. Partially beneficial effects were also observed when agents such as dexamethasone and pentoxiphylline blocking TNF production by macrophages were used.
It is also known that also other cytokines contribute to septic shock. In this situation, the treatments that modulate the cytokine cascade in septic shock have the potential to interfere with the infection containment, since the host defense is dependent upon these same inflammatory cytokines. Finding the means for prevention of septic shock is of a top priority because of potential benefit to a large number of patients. Controlling the level of TNF seems to be essential for these purposes.
Similarly critical is the role of TNF in another defensive mechanism being a fever, which is a physiologic response to infection typical for virtually all higher animals and humans. Five pyrogenic cytokines (interleukin-1, TNF, interferon, interleukin-2 and interleukin-6) are currently recognized as the principal endogenous mediators of the febrile response, inhibiting preoptic warm-sensitive neurons that normally facilitate heat loss and suppress heat production in the human organism. Fever and its mediators have the capacity to harm both the invading organism and the host. Considerable data have been accumulated in recent years suggesting that interleukin-1, TNF and interleukin-6 mediate the pathophysiologic abnormalities of infections. Since the endogenous pyrogens contribute to the pathologic process of various infections both the mediators and febrile response are potentially deleterious to the host. The most convincing evidence in this regard has come from the studies of gram-negative sepsis. There is also the evidence that endogenous pyrogens mediate systemic and local manifestations of sepsis due to gram-positive bacteria, AIDS, spirochetal infections, meningitis, adult respiratory distress syndrome suppurative arthritis and mycobacteriosis. Although the cited data are in contrast to the observation that the febrile response itself heightens resistance to infection in experimental animals, nevertheless, preservation of the species, more than survival of the individual, is the essence of the evolutionary process. Conceivably, the deleterious systemic effects of pyrogenic cytokines on the outcome of overwhelming infections (e.g. gram-negative sepsis) are adapted as beneficial local effects of fever in less fulminant infections. Therefore by hastening the demise of hopelessly infected individuals, nature kills the individuals that are dangerous for the species. In such a way the species as a whole might be protected from epidemic diseases (see Mackowiak, P. A.: Mechanism of Fever; Current Opinions in Infectious Diseases, 1992, 5:348-354).
A fundamental concept of the pathogens of fever is that exogenous pyrogens, regardless of their origin or structure, cause fever by inducing host cells (primarily macrophages) to produce endogenous pyrogens. Accordingly, therapeutic methods based on the use of anti-endogenous pyrogen antibodies and endogenous pyrogen receptor antagonists may be effective. One of the possibilities is the blocking of biosynthesis of TNF. Studies in animals show that TNF might be produced before IL-1 and other cytokines in the cascade of response to infection. According to many scientists, inhibiting the biosynthesis of TNF also means stopping the biosynthesis of IL-1. But inhibiting the biosynthesis of TNF also means stopping the deleterious effects of some fulminant and hopeless infections.
TNF is also known to be one of the mediators of the inflammation processes. The inflammation in many instances is the first stage of a disease in the natural course of which a septic shock develops. In the situation where the continuity of tissues is broken, such as in wounds susceptible to infection, war-like wounds, especially abdominal wounds (peritonitis), diseases in the gastro-intestinal tract such as acute infections accompanying appendicitis, acute bacterial and viral infections as those seen in post-influenza pneumonia, neoplasmic diseases, especially in the phase of decomposition of tumors and the like, inflammation is a first symptom of the increase of TNF production. Controlling the TNF level therefore would be a desired treatment of such infections.
Even more sound is the role of TNF in AIDS itself. AIDS is characterized by a profound immunodeficiency. The hallmark of AIDS is a decreased number of CD4+ lymphocytes. The number of cells infected with HIV, the etiologic agent of AIDS--is relatively small (.ltoreq.1 in 100-1000) even in the peripheral blood mononuclear cells (PBMC) of AIDS patients. While CD4+ lymphocytes are preferentially infected, these cells are not the exclusive targets of HIV infection. Recent evidence has shown that the spectrum of HIV target cells may be quite broad. Clear differences have been observed in the outcome of HIV infection in monocytes/macrophages versus T-lymphocytes. While the T-lymphocytes tend to be destroyed, monocytes/macrophages permit a persistent infection. HIV can therefore be harbored as reservoirs by monocyte/macrophages as well as other cells in the body. The monocyte/macrophage type of response to HIV infection could be responsible for established latency in the host; this response may also cause pathogenic sequelae resulting from soluble factors produced by the infected cells (see Toshifumi Matsuyama et al.: Cytokines and HIV infection: Is AIDS a Tumor Necrosis Factor disease?; AIDS 1991, 5:1405-1417). It was reported by many scientists that human T-cell lines infected with HTLV-1 are highly susceptible to HIV infection, demonstrating dramatic cytopathic effect in association with enhanced replication of HIV. In addition HIV infected cells are susceptible to damage by the supernatant of these cells. Assaying the viral titer after treatment with this supernatant revealed that the factor produced by T-cells (MT-2) enhanced the replication of HIV. The factor was identified as TNF-.beta. and this finding is consistent with reports that T-cells (MT-2) produce TNF-.beta.. The same effect was observed when using TNF-.alpha.. TNF-.alpha. and TNF-.beta. enhanced the replication of HIV. It was also reported that HIV-infected T-cell lines and freshly isolated PBMC from HIV-infected individuals responded to TNF resulting in elevated levels. This suggests that the same enhancement of HIV expression is likely to occur in vivo. In fact, the enhancing activity of TNF could be neutralized by anti-TNF antibodies. The enhancement of HIV replication after treatment with TNF-.alpha. and TNF-.alpha. is up to 10 fold (see Vyakarnam, A. et al.: Tumor necrosis factors (.alpha.,.beta.) induced by HIV-1 in peripheral blood mononuclear cells potentiate virus replication; AIDS 1990, 421-427).
It has also been confirmed that various cytokines can affect HIV production. By employing purified mononuclear phagocytes from normal peripheral blood, both IL-6 and TNF-.alpha. induction was observed within a few hours after exposure to HIV virus. This cytokine induction was also observed using heat-in-activated HIV. Based on many observations Toshifumi Matsuyama et al. (op.cit.) are convinced that AIDS represents a cytokine or TNF disease. In the cytokine network of AIDS, TNF-.alpha. and TNF-.beta. appear to be crucial molecules enhancing the replication of HIV as well as inducing their own expression and that of other cytokines. TNF-.alpha. has been demonstrated to stimulate the release of other cytokines in various cell types, and is therefore to be a key cytokine of the cytokine cascade in the first defence mechanism.
It has been suggested that many of the symptoms associated with AIDS can be explained by the release of cytokines of different biological functions. Enhanced production of IL-1 and TNF-.alpha., the two well known pyrogens could explain fever seen in AIDS patients. TNF-.alpha. may be involved in AIDS-associated cachexia. Both TNF-.alpha. and TNF-.beta. work as immuno-modulators and effector molecules in monocyte-mediated cytotoxicity. Furthermore TNF is responsible for activation of immune response and can directly kill HIV-infected cells, thus enhancing replication of HIV. Also an immunologic mechanism has been proposed to explain CD4-T cell depletion in AIDS (Matsuyama et al., op.cit.). It is also reported that AIDS-related Kaposi's sarcoma is also induced by TNF-.alpha.:TNF-.beta. can be produced from keratinocytes by physiological stimuli such as ultraviolet light, which may contribute to the induction of IL-6 in the skin and development of Kaposi's sarcoma in AIDS. In tests in vitro, TNF-.alpha. can damage myelin and oligodendrocytes; also some glioma-derived cell lines have been shown to be susceptible to the anti-proliferative effect of TNF-.alpha.. This may lead to the conclusion that the dysfunction of the central nervous system in AIDS patients is a result of TNF-.alpha. involvement. Several reports have shown that the serum levels of TNF-.alpha. and IL-1 are substantially elevated with the development of AIDS and ARC (AIDS related complex) while they fell within the range of healthy control values in tests of serum of asymptomatic carriers of HIV. According to Matsuyama et al. (op.cit.), IDS is as much a TNF disease as it is a HIV disease. It shows that gaining a control over TNF induction may lead to establishing effective therapy for AIDS patients.